DyNA Quant 120 Capillary Adaptor Kit
Contents
1. Important All solutions to be measured must con tain enough 10X TNE buffer stock so that the final concentration is no less than 1X TNE Prepare all solutions in microfuge tubes with lids Keep lid closed between measurements to prevent evapora tion Capillary DNA range DNA solution CAS size ul ng ul pl 10 25 1500 5 5 50 10 1500 45 5 100 10 1500 95 5 p 6 DyNA Quant 120 Example This example is for preparing solutions for 50 pl capillary tubes using a standard containing 1000 ng DNA in 50 ul Blank 45 ul 1X TNE buffer 5 ul CAS Standard 45 ul DNA 5 ul CAS for 1000 ng total DNA in 45 ul final 10 ul 100 ng ul DNA 35 ul 1X TNE Sample 45 ul DNA sample in 1X TNE 5 ul CAS Zero the instrument 1 Filla capillary with blank solution Insert the capil lary tube into the sample solution and allow the liquid to almost fill the tube Tilt the larger 50 and 100 pl tubes into a horizontal position if necessary 2 Seal the capillary tube Hold the sealant pad vertically press the tube no more than 2 mm into the sealant and twist gently Wipe the tube with tissue and inspect the seal for leaks 3 Insert the unsealed end of the blank into the adaptor As you place the capillary and adaptor in the cuvette well aim the capillary end of the assembly so that it fits into the depression in the bottom of the well Gently push the adaptor into place Note The sealant which fluoresces must be completel2. mix the sample and assay solution Use a micropipet accurate to at least 0 02 pl If inconsis tencies persist dilute the sample and use larger aliquots Take care that the DNA sample is completely suspend ed Use only pure distilled and filtered 0 2 or 0 4 um filter water for all solutions Filter the 1X TNE buffer to remove all particulates Particulates may cause light to scatter causing measure ment fluctuations Filter the buffer before adding Hoechst dye because the dye binds to most membrane types Use as little sealing compound as possible Sealant is flu orescent and will cause erroneous readings if it enters the light path Wipe the capillary to remove fingerprints and liquids Readings A J Vv negative or lower than expected Use freshly prepared assay solution at ambient temper ature to set the zero and for all subsequent measure ments Extract ethidium bromide from DNA solutions because it interferes with the fluorescence of Hoechst dye If expected DNA values are based on A269 A2g read ings the sample may be contaminated with RNA nucleotides or protein which are not detected by H 33258 fluorescence Crude cell lysates prepared with acid guanidinium thiocyanate phenol solution y Use Readings J Fluorescence of lysates prepared without an alkaline EDTA pretreatment is reduced by 70 compared to lysates with such pretreatment Alkaline conditions allow formation of comple
3. adaptor which fits most fluo rometers one scoring file one bulb assembly and silicone grommet to hold the capillary and one pad of sealing compound Unpacking Unwrap all packages carefully and compare contents with the packing list making sure all items arrived If any part is missing contact your local sales office Inspect all com ponents for damage that may have occurred in transit If any part appears damaged contact the carrier immediate ly Be sure to keep all packing material for damage claims or for repacking should it become necessary to return an item Capilla tube idant Bulb assembly and grommet to api ary TUDE AGAPE N expel sample after measurement Capillary tubes Sealing compound a d DyNA Quant 120 ep1 p 2 DyNA Quant 120 Important Hoechst 33258 dye is a possible carcinogen and possi ble mutagen Wear gloves when handling Wear a mask when weigh ing Disposal must comply with all applicable regula tions Never dispose of by pouring into a drain b Because these measurements are highly sensitive take extra steps to assure clean materials and reagents Use sterile pipette tips and sample tubes Make TNE buffer with double distilled sterile water Filter buffers and water before adding H 33258 Use only highly pure chemicals Wear gloves Fluorescence measurement DyNA Quant 200 Bisbenzimide commonly known as Hoechst 33258 H 33258 dye exhibits changes in fluorescenc
4. dard curve See the DyNA Quant 200 Instructions Section 4 Analyze the results for more detail To recover the sample for electrophoresis 1 es Remove the capillary from the adaptor and fit the bulb on the capillary taking care to not cover the hole at the top of the bulb Score the capillary tube with the file just above the seal ing compound plug and carefully snap this section off Cover the opening in the bulb with your fingertip and squeeze gently to expel the sample into a tube DyNA Quant 120 ep 9 p10 DyNA Quant 120 Troubleshooting Always be sure to A y s Operate the unit in a location isolated from equipment that radiates high frequency electromagnetic interfer ence Operate the unit away from direct sunlight Place the instrument so that the back vents are not obstructed Fluorescence values drift s S Assay solutions must be at ambient temperature for consistent readings Fluorescence decreases as tempera ture increases Protect fluorescent reagents and samples from light to prevent photobleaching destruction of the fluorescent compound by light Take readings immediately to prevent photobleaching Assay solutions must be at pH 7 4 Adjust the salt concentration For standard DNA the concentration should be at least 200 mM NaCl in 1X TNE For crude cell lysates use 2 to 3 M NaCl in 1X TNE Wide fluctuations in fluorescence values Se RAS RES Thoroughly
5. e characteris tics in the presence of DNA that allow accurate DNA quantitation In the absence of DNA the fluorescence excitation spectrum of H 33258 peaks at 356 nm and the emission spectrum peaks weakly at 492 nm When H 33258 binds to DNA these peaks shift to 365 nm ex and 458 nm em In the cuvette well the sample is exposed to filtered light 365 7 nm from a mercury lamp This light excites the DNA dye complex causing light that peaks at 458 nm to be emitted An emission filter in front of the the photodetector allows only fluorescence at 460 nm 15 nm to register Thus the measured fluorescence is a direct indicator of the DNA concentration Measurement considerations Dilute DNA standard and sample in 1X TE to ensure the optimal pH value 7 4 and maximum signal to noise ratio The dye concentration for the capillary adaptor assay is necessarily higher than the dye concentration for the 2 ml cuvette assay because the same amount of DNA is in a much smaller volume of assay solution Enough dye must be present to preserve the linear relationship between the amount of DNA and the fluorescence measured Note Using smaller volumes does not increase the sensi tivity of the assay Note The blank standard and sample solutions all must contain the same final dye concentration at the same pH and all must have similar ionic strength The DNA reference standard 1 mg ml solution of calf thymus DNA serves both to provide a one poin
6. es containing up to 1500 ng total DNA First mix the desired amount of DNA with 1X TNE to a volume of 45 pl Use a 1 10 dilution of the 1 mg ml DNA standard for a 100 ng l working solution Then add 5 pl capil lary assay solution Note For multiple measurements multiply each amount by the number of readings desired Desired ng DNA per tube 0 300 600 90012001500 ul DNA solution 0 3 6 9 12 15 100 ng ul 1 10 dilution 1X TNE to 45 ul total 45 42 39 36 33 30 Capillary assay solution ul 5 5 5 5 5 5 2 es Measure a sample for each concentration If desired measure a second sample and average the readings Plot the data or enter it into a math program to find the linear best fit equation of the line passing through the points See the DyNA Quant 200 instructions Section 4 Analyze the results for more detail Measure unknown DNA concentration 1 2 es Fill the capillary with DNA sample solution and seal Insert the capillary into the adaptor and place the adap tor into the cuvette well Close the lid and record the reading Depending on how the instrument was cali brated the display shows either the actual DNA con centration or a multiple of the concentration If the reading is above the linear range of the concen tration curve use less DNA and repeat the assay Determine the concentration from the standard concen tration curve or calculate the concentration from the equation of the stan
7. filter Store in an amber bottle at 4 C for up to 6 months 1M Tris Cl pH 7 4 100 ml Tris base Tris hydroxymethyl aminomethanel MW 121 14 12 11 g dd H20 80 ml Adjust pH to7 4 with concentrated HCI Let solution cool to room temperature before final addition of HCI to pH 8 0 Adjust final volume to 100 ml with distilled water Autoclave 5M NaCl 100 ml NaCl 29 22 g Add distilled water to 100 ml and autoclave 0 5M EDTA disodium ethylenediaminetetracetate pH 8 0 100 ml EDTA 18 61 g dd H20 80 ml Stir vigorously with magnetic stir bar Titrate pH to approximately 8 0 with NaOH Adjust final volume to 100 ml with distilled water Autoclave 1X TE buffer stock solution 10 mM Tris Cl 1 mM EDTA pH 7 4 100 ml 1 M Tris Cl pH 7 4 1 mL 0 5 M EDTA pH 8 0 0 2 mL dd H20 98 8 mL Adjust pH to 7 4 with concentrated HCI Autoclave and store at room temperature for up to 3 months 10X TNE buffer stock solution 100 mM Tris 10 mM EDTA 2 M NaCl 100 ml 1M Tris Cl pH 7 4 10 ml 0 5 M EDTA pH 8 0 2 ml 5M Nacl 40 ml Add distilled water to 100 ml Adjust pH to 7 4 with concentrat ed HCl Autoclave and store at 4 C for up to 3 months Calf thymus DNA 100 pg ml 1 10 dilution of standard may require further dilution 100 ul calf thymus DNA standard 1 mg ml 100 pl 10X TNE 800 ul distilled water filtered Gently tap the tube to mix thoroughly Store at 4 C for up to 3 months 2X Capillary assay solut
8. fus r manual DNA Protein Labeling Hybridization amp Detection DyNA Quant 120 Capillary Adaptor Kit an accessory to the DyNA Quant 200 Amersham im 80 6232 00 DQ120 IM Rev B0 8 99 e Y Biosciences e DyNA Quant 120 contents Kit FUNCTION sa ciicvecsivivcetecevakcencrentneieveelcivacduaveviievend cy 1 UMD OCKING neriesi Garey decratbendinn desdenatencess core 1 Fluorescence measurement ssssssssssssnrnnnnnnnnnnnnnnnnnnnnn 2 Measurement Considerations succeer 2 Method OVErViCW ccccccececcseeeeeeeseseeeeuseevessneenesaess 3 SOLUTIONS aeina 4 Operating Instructions vices eerie 6 SetuP antaen ata i a tateret eee gone 7 Zero the NSCFUMOENC eis sccec lvveccesvasdcodvactuetvvcdves beds 7 Set a reference point with DNA standard 8 Construct a standard CUrVE cccccccseceeeeee esse seen ees 8 Measure unknown DNA concentration 005 9 Troubleshooting wc 10 Customer Service Information scsccceceseersreeeeeee 13 Technical Service and repair cceeeeeeeeeeeeeees 13 Ordering information ccccceceeeeeeeeeeeeeeeenaee 13 Capillary Adaptor Kit Function Use the DyNAQuant 120 Capillary Adaptor Kit for microvolume 10 100 ul quantitation of DNA or enzyme activity With this adaptor sample can be easily recovered for further processing Capillaries are disposable and thus eliminate cross contamination The kit includes 20 each of 10 50 and 100 pl capillary tubes one capillary tube
9. ion 20 ug ml H 33258 in 1X TNE pH 7 4 H 33258 stock solution 20 ul 10X TNE buffer stock solution 100 ul Distilled filtered water 880 ul Prepare fresh daily Keep at room temperature Do not filter once the dye is added DyNA Quant 120 ep5 Operating instructions Setup Since the DNA fluorescence assay is based on a relative measurement of emitted light a calibration reference value must be established with a known DNA sample before the concentration of DNA in unknown samples can be deter mined Once the initial reference value can be reliably reproduced you can proceed to determine the concentra tions of unknown samples or determine assay linearity with standard concentration measurements Generating a standard curve once every few weeks serves as a quality check on the standard a reliability check on the instru ment and a consistency check on technique 1 Turn on the fluorometer lamp at least 15 minutes before taking measurements so that the lamp has time to stabi lize Note Switch the prompt off when measuring fluorescence with the capillary adaptor Select 2 gt Setup 1 gt Prompt and select 1 gt Off 2 Prepare stock solutions 3 Select a capillary size according the desired DNA sam ple volume see table below You will need one blank to zero the instrument one standard solution to cali brate the instrument optional plus a series of standard concentrations for a standard curve and DNA sam ples
10. it please call or fax your local Amersham Pharmacia Biotech representative Important Request a copy of the Amersham Pharmacia Biotech Contamination Clearance Certificate before returning the item No items can be accepted for servicing or return unless this form is properly completed Ordering Information Basic Unit DyNA Quant 200 Fluorometer 1 80 6406 80 Includes DNA standard Hoechst 33258 dye 100 mg and Performance Validation Kit 115 230 V Glass fluorometry cuvette 1 80 6227 44 fluorescent grade Lamp replacement assembly 1 80 6228 96 Optics replacement kit Includes filter glass cover mirrors and O ring 1 80 6229 34 Lid replacement assembly Includes lid latch 1 80 6229 53 spring and mounting screw Capillary Adaptor Capillary Adaptor Kit 1 80 6227 63 includes capillary tubes 10 50 and 100 ul 20 each Capillary tubes 10 ul 100 80 6227 82 Capillary tubes 50 ul 100 80 6228 20 Capillary tubes 100 ul 100 80 6228 01 Dye and standards Hoechst 33258 dye 100 mg 1 80 6226 87 Calf thymus DNA standard 250 ug 80 6227 06 4 methylumbelliferone standard 100 mg 1 80 6227 25 Performance Validation Kit 1 80 6252 52 DyNA Quant 120 ep 13 Printed in the USA Amersham Biosciences UK Limited Amersham Place Little Chalfont Bucks HP7 9NA England Amersham Biosciences SE 751 84 Uppsala Sweden Amersham Biosciences 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Eur
11. ope GmbH Postfach 5480 9 D 79021 Freiburg DyNA Quant are trademarks of Amersham Biosciences Limited or its subsidiaries Amersham Biosciences is a trademark of Amersham plc Tris is a trademark of Union Carbide Chemicals and Plastics Co All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group which sup plies them A copy of these terms and conditions is available on request Amersham Biosciences 1999 All rights reserved Amersham e Y Biosciences
12. t refer ence for determining the DNA concentration of unknown DNA samples and as a standard to evaluate the perfor mance of the fluorometer Generate a standard concentration curve for maximum accuracy Analyse the results by graphing the sample con centration x vs the averaged reading y As long as the graph is linear you can expect accurate values for unknown samples within the range of the standard curve Slight variations are most commonly due to pipetting variability Method overview 1 Turn on the fluorometer lamp at least 15 minutes before using 2 Prepare stock solutions Prepare all blank standard and sample solutions in microfuge tubes with lids 3 Zero and calibrate the instrument Optional Measure a series of standard concentrations and plot a concentration curve that spans the expected sample concentration range Analyze the results by graphing or determining the least squares fit equation of the line 4 Measure the fluorescence of the unknown DNA sam ple If you constructed a concentration curve correlate the measurements to the curve DyNA Quant 120 ep 3 Note Disodium EDTA does not go into solu tion until pH approaches 8 0 p 4 DyNA Quant 120 Solutions Important Refer to the material safety data sheet MSDS accompanying each chemical for detailed han dling and safety information Hoechst 33258 stock solution 1mg ml Hoechst 33258 10 mg Distilled filtered water 10 mi Do not
13. xes between DNA and the dye For a detailed protocol see Rymaszewski et al 1990 Estimation of cellular DNA content in cell lysates suitable for RNA isolation Anal Biochem 188 91 96 the appropriate reference standard Use an ultra pure grade DNA standard Make sure to use a standard with a G C content very similar to the sample DNA Hoechst dye binds prefer entially to A T regions so G C content must be similar to ensure equivalent binding Use a ssDNA standard for ssDNA samples Single stranded DNA yields about half the fluorescence of an equal amount of double stranded DNA Plasmid DNA standards should have the same confor mation as the sample Each form supercoiled relaxed circular or linear may have slightly different dye bind ing characteristics higher than expected Fluorescence enhancement may result from high levels of detergents Final SDS concentration should be below 0 01 and other detergents below 10 pg ml the final concentration of any detergent should be well below its critical micelle concentration Use a reference standard with a G C content very simi lar to your sample Single stranded genomic DNA yields about half the flu DyNA Quant 120 e p 11 Customer Service Information Technical Service and Repair Amersham Pharmacia Biotech offers complete technical support for all our products If you have any questions about how to use this product or would like to arrange to repair
14. y seated in the depression to avoid erroneous readings 4 Close the lid and press lt ZERO gt Capillary position before and after installing adaptor into the cuvette Adaptor placement well DyNA Quant 120 ep7 p 8 DyNA Quant 120 Set a reference point with DNA standard 1 Filla capillary with the appropriate DNA standard We suggest using a DNA concentration higher than the expected concentration of the unknown sample Seal the capillary tube as in step 2 above 2 Insert the capillary into the adaptor position the adap tor in the cuvette well close the lid and press lt CALIB gt Enter the actual concentration of the standard or enter a convenient value that will display a multiple of the actual DNA concentration Press lt ENTER gt Example If your standard contains 100 ng DNA and you enter 25 each unit then corresponds to 0 25 ng DNA in the capillary and the value displayed must be multiplied by 4 es If desired measure a second capillary containing a stan dard solution to verify that results are reproducible One reference point is adequate to set the instrument However generating a standard concentration curve assures assay linearity in the range of interest An example of setting up a standard curve with a DNA range of 0 to 1500 ng for a 50 pl capillary tube follows on page 9 Construct a standard curve 1 Prepare a set of standards The following example applies to 50 pl capillary tub
Download Pdf Manuals
Related Search